The present invention relates to recombinant DNA molecules which encode bovine respiratory syncytial (BRS) virus proteins as well as corresponding BRS virus proteins and peptides derived therefrom. It is based, in part, on the cloning of full length cDNAs encoding a number of bovine respiratory syncytial virus proteins, including, F, G, and N. DNAs encoding the G and F proteins have been inserted into vaccinia virus vectors, and these vectors have been used to express the G and F proteins in culture and G protein encoding vectors have been used to induce an anti-bovine respiratory syncytial virus immune response. The molecules of the invention may be used to produce safe and effective bovine respiratory syncytial virus vaccines.
Bovine respiratory syncytial (BRS) virus strain 391-2 was isolated from an outbreak of respiratory syncytial virus in cattle in North Carolina during the winter of 1984 to 1985. The outbreak involved five dairy herds, a beef calf and cow operation, and a dairy and steer feeder operation (Fetrow et al., North Carolina State University Agric. Extension Service Vet. Newsl.).
Respiratory syncytial virus, an enveloped, single-stranded, negative-sense RNA virus (Huang and Wertz, 1982, J. Virol. 43:150-157; Kingsbury et al., 1978, Intervirology 10:137-153), was originally isolated from a chimpanzee (Morris, et al., 1956, Proc. Soc. Exp. Biol. Med. 92:544-549). Subsequently, respiratory syncytial virus has been isolated from humans, cattle, sheep and goats (Chanock et al. 1957, Am. J. Hyg. 66:281-290; Evermann et al., 1985, AM. J. Vet. Res. 46:947-951; Lehmkuhl et al., 1980, Arch. Virol. 65:269-276; Lewis, F. A., et al., 1961, Med. J. Aust. 48:932-33; Paccaud and Jacquier, 1970, Arch. Gesamte Virusforsch 30:327-342). Human respiratory syncytial (HRS) virus is a major cause of severe lower respiratory tract infections in children during their first year of life, and epidemics occur annually (Stott and Taylor, 1985, Arch. Virol. 84:1-52). Similarly, BRS virus causes bronchiolitis and pneumonia in cattle, and there are annual winter epidemics of economic significance to the beef industry (Bohlender et al., 1982, Mod. Vet. Pract. 63:613-618; Stott and Taylor, 1985, Arch. Virol. 84:1-52; Stott et al., 1980, J. Hyg. 85:257-270). The highest incidence of severe BRS virus-caused disease is usually in cattle between 2 and 4.5 months old. The outbreak of BRS virus strain 391-2 was atypical in that the majority of adult cows were affected, resulting in a 50% drop in milk production for one dairy herd and causing the death of some animals, while the young of the herds were only mildly affected (Fetrow et al., 1985, North Carolina State University Agric. Extension Service Vet. Newsl.).
BRS virus was first isolated in 1970 (Paccaud and Jacquier, 1970, Arch. Gesamte Virusforsch. 30:327-342), and research has focused on the clinical (van Nieuwstadt, A. P. et al., 1982, Proc. 12th World Congr. Dis. Cattle 1:124-130; Verhoeff et al., 1984, Vet. Rec. 114:288-293) and pathological effects of the viral infection on the host (Baker et al., 1986, J. Am. Vet. Med. Assoc. 189:66-70; Castleman et al., 1985, Am. J. Vet. Res. 46:554-560; Castleman et al. 1985, Am. J. Vet. Res. 46:547-553) and on serological studies (Baker et al., 1985, Am. J. Vet. Res. 46:891-892; Kimman et al., 1987, J. Clin. Microbiol. 25:1097-1106; Stott et al., 1980, J. Hyg. 85:257-270). The virus has not been described in molecular detail. Only one study has compared the proteins found in BRS virus-infected cells with the proteins found in HRS virus-infected cells (Cash et. al., 1977, Virology 82:369-379). In contrast, a detailed molecular analysis of HRS virus has been undertaken. cDNA clones to the HRS virus mRNAs have been prepared and used to identify 10 virus-specific mRNAs which code for 10 unique polypeptides, and the complete nucleotide sequences for 9 of the 10 genes are available (Collins, P. L., et al., 1986, in xe2x80x9cConcepts in Viral Pathogenesis II,xe2x80x9d Springer-Verlag., New York; Stott and Taylor, 1985, Arch. Virol. 84:1-52).
Two lines of evidence suggest that HRS virus and BRS virus belong in distinct respiratory syncytial virus subgroups. First, BRS virus and HRS virus differ in their abilities to infect tissue culture cells of different species (Paccaud and Jacquier, 1970, Arch. Gesamte Virusforsch. 30:327-342). With one exception, studies have shown that BRS virus exhibits a narrower host range than HRS virus. Matumoto et al. (1974, Arch. Gesamte Virusforsch. 44:280-290) reported that the NMK7 strain of BRS virus has a larger host range than the Long strain of HRS virus. Others have been unable to repeat this with other BRS strains (Paccaud and Jacquier, 1970, Arch. Gesamte Virusforsch. 30:327-342; Pringle and Crass, 1978, Nature (London) 276:501-502). The second line of evidence indicating that BRS virus differs from HRS virus comes from the demonstration of antigenic differences in the major glycoprotein, G, of BRS virus and HRS virus (Orvell et al., 1987, J. Gen. Virol. 68:3125-3135). Studies using monoclonal antibodies have grouped HRS virus strains into two subgroups on the basis of relatedness of the G glycoprotein (Anderson 1985, J. Infect. Dis. 151:626-633; Mufson, et al., 1985, J. Gen. Virol. 66:2111-2124). The G protein of BRS virus strains included in these studies did not react with monoclonal antibodies generated against viruses from either HRS virus subgroup (Orvell et al., 1987, J. Gen. Virol. 68:3125-3135).
BRS virus provides an opportunity to study the role of the major glycoprotein, G, in attachment, the possible host range restrictions of BRS virus compared to HRS virus, and the roles of the individual viral antigens necessary to elicit a protective immune response in the natural host, which is something that cannot be done easily for HRS virus at present.
Previous work has shown that there is no cross reactivity between the attachment surface glycoproteins, G, of BRS virus and HRS virus, whereas there is cross antigenic reactivity between the other transmembrane glycoprotein, the fusion, F, protein and the major structural proteins, N, P, and M (Lerch et al., 1989, J. Virol. 63:833-840; Orville et al., 1987, J. Gen. Virol. 68:3125-3135). Available evidence indicates that BRS virus has a more narrow host restriction, infecting only cattle and bovine cells in culture, whereas HRS virus can infect a variety of cell types and experimental animals (Jacobs and Edington, 1975, Res. Vet. Sci. 18:299-306; Mohanty et al., 1976, J. Inf. Dis. 134:409-413; Paccaud and Jacquier, 1970, Arch. Gesamte Virusforsch 30:327-342). Since the G protein of HRS virus is the viral attachment protein (Levine et al., 1987, J. Gen. Virol. 68:2521-2524), this observation suggested that the differences in the BRS virus and HRS virus G proteins may be responsible for the differences in attachment and host range observed between BRS virus and HRS virus.
Based on sequence analysis of the HRS virus G mRNA, the G protein is proposed to have three domains; an internal or cytoplasmic domain, a transmembrane domain, and an external domain which comprises three quarters of the polypeptide (Satake et al., 1985, Nucl. Acids Res: 13:7795-7812; Wertz et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:4075-4079). Evidence suggests that the respiratory syncytial virus G protein is oriented with its amino terminus internal, and its carboxy terminus external, to the virion (Olmsted et al., 1989, J. Viral. 13:7795-7812; Vijaya et al., 1988, Mol. Cell. Biol. 8:1709-1714; Wertz et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:4075-4079). Unlike the other Paramyxovirus attachment proteins, the respiratory syncytial virus G protein lacks both neuraminidase and hemagglutinating activity (Gruber and Levine, 1983, J. Gen. Virol. 64:825-832; Richman et al., 1971, Appl. Microbiol. 21:1099). The mature G protein, found in virions and infected cells, has an estimated molecular weight of 80-90 kDa based on migration in SDS-polyacrylamide gels (Dubovi, 1982, J. Viol. 42:372-378; Gruber and Levine, 1983, J. Gen. Virol. 64:825-832; Lambert and Pons, 1983, Virology 130:204-214; Peeples and Levine, 1979, Viol. 95:137-145). In contrast, the G mRNA sequence predicts a protein with a molecular weight of 32 kDa (Satake et al., 1985, Nucl. Acids Res. 13:7795-7812; Wertz et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:4075-4079), and when the G mRNA is translated in vitro it directs synthesis of a 36 kDa protein that is specifically immunoprecipitated by anti-G serum. It has been shown that there is N-linked and extensive O-linked glycosylation of the polypeptide backbone (Lambert, 1988, Virology 164:458-466; Wertz et al., 1989, J. Virol. 63:X). Experiments using glycosidases (inhibitors of sugar addition) and a cell line defective in O-linked glycosylation suggest that 55% of the molecular weight of the mature G protein is due to O-linked glycosylation, and 3% is due to N-linked glycosylation. However, these estimates are based on migration in SDS-polyacrylamide gels and are only approximate values. Consistent with the evidence for extensive O-linked glycosylation is a high content (30%) of threonine and serine residues in the predicted amino acid sequence of the G protein (Satake et al., 1985, Nucl. Acids Res. 13:7795-7812; Wertz et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:4075-4079). Threonine and serine are amino acid residues that serve as sites for O-linked oligosaccharide attachment (Kornfield and Kornfield, 1980, in xe2x80x9cThe Biochemistry of Glycoproteins and Proteoglycans,xe2x80x9d Lenarz, ed., Plenum Press, N.Y. pp. 1-32) and in the HRS virus G protein 85% of the threonine and serine residues are in the extracellular domain (Wertz et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:4075-4079). The high content of proline (10%), serine and threonine (30%) and the extensive O-linked glycosylation of the G protein are features similar to those of a group of cellular glycoproteins known as the mucinous proteins (Ibid.), but unusual among viral glycoproteins.
Isolates of HRS virus have been divided into two subgroups, A and B, based on the antigenic variation observed among G proteins using panels of monoclonal antibodies (Anderson et al., 1985, J. Inf. Dis. 151:626-633; Mufson et al., 1985, J. Gen. Virol. 66:2111-2124). However, a few monoclonal antibodies exist which recognize the G protein of both subgroups (Mufson et al., 1985, J. Gen. Virol. 66:2111-2124, orvell et al., 1987, J. Gen. Virol. 68:3125-3135). Sequence analysis of the G mRNA of HRS viruses from the two subgroups showed a 54% overall amino acid identity between the predicted G proteins, with 44% amino acid identity in the extracellular domain of the protein (Johnson et al., 1987 Proc. Natl. Acad. Sci. U.S.A. 84:5625-5629).
The mature BRS virus F protein consists of two disulfide linked polypeptides, F1 and F2 (Lerch et al., 1989, J. Virol. 63:833-840). There are differences in the electrophoretic mobility of the BRS virus and HRS virus F protein in SDS-polyacrylamide gels (Lerch et al., 1989, supra). Polyclonal and most monoclonal antibodies react to the F protein of both HRS virus and BRS virus (Stott et al., 1984; Orvell et al., 1987; Kennedy et al., 1988, J. Gen. Virol. 69:3023-3032; Lerch et al., 1989, supra).
The fusion protein, F, of paramyxoviruses causes fusion of the virus to cells and fusion of infected cells to surrounding cells. Structurally, the F proteins of the various paramyxoviruses are similar to one another. The F protein is synthesized as a precursor, F0, that is proteolytically cleaved at an internal hydrophobic region to yield two polypeptides, F1 and F2, that are disulfide linked and form the active fusion protein. A carboxy terminal hydrophobic region in the F1 polypeptide is thought to anchor the F protein in the membrane with its carboxy terminus internal to the cell and the amino terminus external. The F protein is N-glycosylated (see review, Morrison, 1988, Virus Research 10:113-136). The HRS virus F protein is a typical paramyxovirus fusion protein. Antibodies specific to the F protein will block the fusion of infected cells (Walsh and Hruska, 1983, J. Virol. 47:171-177; Wertz et al., 1987, J. Virol. 61:293-301) and also neutralize infectivity of the virus (Fernie and Gerin, 1982, Inf. Immun. 37:243-249; Walsh and Hruska, 1983, J. Virol. 47:171-177; Wertz et al., 1987, J. Virol. 61:293-301), but do not block attachment (Levine et al., 1987, J. Gen. Virol. 68:2521-2524). The F protein is synthesized as a polypeptide precursor F0, that is cleaved into two polypeptides, F1 and F2. These two polypeptides are disulfide linked and N-glycosylated (Fernie and Gerin, 1982, Inf. Immun. 37:243-249; Gruber and Levine, 1983, J. Gen. Virol. 64:825-832; Lambert and Pons, 1983, Viral. 130:204-24).
Bovine respiratory syncytial virus (BRS) vaccines have been developed comprising live or inactivated virus, or viral proteins. Frennet et al. (1984, Ann. Med. Vet. 128:375-383) reported that 81 percent of calves administered a combined live BRS virus and bovine viral diarrhea vaccine were protected against severe respiratory symptoms induced by field challenge. Stott et al. (1984, J. Hyg. 93:251-262) compared an inactivated BRS viral vaccine (consisting of glutaraldehyde-fixed bovine nasal mucosa cells persistently infected with BRS virus and emulsified with oil adjuvant) to two live vaccines, one directed toward BRS virus and the other toward HRS virus. Eleven out of twelve calves given the inactivated viral vaccine in the Stott study (supra) were completely protected against BRS viral challenge, but all control animals and those given the live vaccines became infected.
It is possible that live vaccines may exacerbate BRS viral infection. A severe outbreak of respiratory disease associated with BRS virus occurred shortly after calves were vaccinated with a modified live BRS virus (Kimman et al., 1989, Vet. Q. 11:250-253). Park et al. (1989, Res. Rep. Rural Dev. Adm. 31:24-29) reports the development of a binary ethylenimine (BEI)-inactivated BRS virus vaccine which was tested for its immunogenicity in guinea pigs and goats. Serum neutralizing antibody was detected 2 weeks following inoculation and antibodies increased following a booster vaccination at four weeks. In goats, a protective effect against BRS virus was observed when animals were challenged with virus 12 weeks following inoculation.
Trudel et al. (1989, Vaccine 7:12-16) studied the ability of immunostimulating complexes, made from the surface proteins of both human (Long) and bovine (A-51908) RS strains, adsorbed to the adjuvant Quil A, to induce neutralizing antibodies. Immunostimulating complexes prepared from bovine RS virus proteins were found to be significantly more efficient than their human counterpart in inducing neutralizing antibodies.
The present invention relates to recombinant DNA molecules which encode bovine respiratory syncytial (BRS) virus proteins, to BRS virus proteins and peptides and to recombinant BRS virus vaccines produced therefrom. It is based, in part, on the cloning of substantially full length cDNAs which encode the entire BRS virus G, F, and N proteins. Nucleotide sequences of the G, F, and N cDNAs have been determined, and are set forth in FIGS. 2A-C (G protein), FIGS. 9A-C (F protein), and FIGS. 17A and B (N protein).
According to particular embodiments of the invention, DNA encoding a BRS virus protein or peptide may be used to diagnose BRS virus infection, or, alternatively, may be inserted into an expression vector, including, but not limited to, vaccinia virus, as well as bacterial, yeast, insect, or other vertebrate vectors. These expression vectors may be utilized to produce the BRS virus protein or peptide in quantity; the resulting substantially pure viral peptide or protein may be incorporated into subunit vaccine formulations or may be used to generate monoclonal or polyclonal antibodies which may be utilized in diagnosis of BRS virus infection or passive immunization. In additional embodiments, BRS virus protein sequence provided by the invention may be used to produce synthetic peptides or proteins which may be utilized in subunit vaccines, or polyclonal or monoclonal antibody production. Alternatively, a nonpathogenic expression vector may itself be utilized as a recombinant virus vaccine.